Granulosa cells in the follicle proliferate and differentiate in concordance with progression of the estrus cycle and pregnancy. The significance of cell cycle control in reproductive tissues is recapitulated by female infertility observed in knockout mice deficient for components of G 1-cell cycle machinery, i.e., cyclin D2, Cdk4 and p27 KiN. The longterm goal of this program is to elucidate the role of the cell cycle-regulatory machinery in the homeostasis of female reproductive function. The objective of this project is to determine how the G1-Cdk pathways control ovarian folliculogenesis and luteinization. Folliculogenesis from the preantral to preovulatory stage is associated with FSH-dependent rapid cell cycle progression of granulosa cells. Previous studies using cyclin D2-null mice showed that this exponential proliferation depends on cyclin D2. In contrast, mice deficient for Cdk4, the major cyclin D2-associated kinase, display normal FSH-dependent proliferation and intact development of preovulatory follicles. These data suggest that cyclin D2 may activate other Cdk(s), possibly Cdk6, in granulosa cells upon FSH and estrogen stimuli. Shortly after the ovulatory LH surge, granulosa cells in preovulatory follicles cease proliferation, accompanied by loss of cyclin D2 expression and upregulation of cyclin D3 and the two Cdk inhibitors, p21Cipl and p27 Kip1. The withdrawal from the cell cycle coincides with differentiation of granulosa cells into highly steroidogenic luteal cells. Ovaries of mice deficient for p27 show a significant delay for luteinized granulosa cells to cease proliferation, which is further enhanced by co-disruption of p21. In this renewal application, we will evaluate the central hypothesis that rapid proliferation of granuiosa cells during folliculogenesis requires cyclin D2-dependent activation of Cdk4 and Cdk6, while the upregulation of cyclin D3, p21 and p27 plays critical roles in establishing quiescence and maintaining viability of differentiated luteal cells.
The specific aims to be pursued are: (1) Determine whether Cdk6 and Cdk4 collaborate with cyclin D2 in the development of preovulatory follicles; (2) Determine the roles of cyclin D2 downregulation and cyclin D3 upregulation in luteinizing and luteinized granulosa cells; (3) Determine how p21Cipl and p27 Kipl control quiescence of luteal cells. These studies will provide significant insight into granulosa/luteal cell-specific roles of the G 1-regulatory proteins. No available cell line would generate such information. It is our expectation that this program contributes to a scientific foundation for therapeutic targets against infertility and other ovarian diseases.
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